Flow cytometry is a powerful method of analysis to determine the cellular/biological content of various types of samples, and in particular samples that contain living cells. In clinical applications, flow cytometers are useful for myriad of applications including lymphocyte counting and classification, for immunological characterization of leukaemias and lymphomas, leukocyte counting and classification, and bead-based immunoassay diagnostics.
In most flow cytometry techniques, cells in a fluid solution are caused to flow individually through a light beam, usually produced by a laser light source or a monochromatically filtered LED source. As light strikes each cell, the light is scattered and the resulting scattered light is analyzed to determine the type of cell. The cell may also optionally be labelled with a marker linked to a fluorescent molecule, which fluoresces when light strikes it and thereby reveals the presence of the marker on the cell. In this fashion, information about the surface components of the cell can be obtained.
Fluorescent beads provide a convenient and powerful method for performing multiplexed immunoassays. For example, multiplexing may be achieved by using beads of different colours (either scattered or fluorescence emission) coupled with analytes that are uniformly dyed with another distinct colour. In this way, the assay identity may be read from the bead colour/fluorescence while the analytic result can be read from the amount of fluorescent dye bound to the bead. Beads also provide a format in which solid phases of immunoassays may be manufactured in bulk, increasing the robustness of assays. Typically, flow cytometers are used to perform multiplexed, bead-based immunoassays, by virtue of their ability to detect and quantify fluorescence from particles.
One significant problem with currently implemented immunofluorescent bead assays is that they require extensive manual or robotic sample processing. For a sandwich-type immunoassay, this sample processing may consist of some or all of the following steps:                separation of plasma from whole blood        metering of plasma to provide known volume of starting sample        dilution of plasma        mixing of dilute sample with immunofluorescent beads to promote binding of analyte        incubation for sufficient time and at a temperature appropriate for binding        washing of beads        mixing of washed beads with labelled secondary antibody to promote binding        incubation for sufficient time and at a temperature appropriate for binding        washing of unbound secondary antibody        injection of beads into flow cytometric flow channel.        
Manual processing is cost-prohibitive in many applications and is also prone to errors. Automation is also cost-prohibitive in many applications, and is inappropriate as currently practiced—using, for example, liquid handling robots—for applications such as point-of-care or doctor's office analysis. As a result, there is an unmet need to provide sample processing for multiplexed, bead-based immunoassays that is less expensive and less prone to error than current automation or manual processing.
A drawback of Point-of-Care diagnostic assay systems is that they are typically incapable of multiplexing a variety of assay types. While these systems are quite good at performing a variety of similar assay types—such as lateral flow assays, or electrochemical assays, etc.—the assay conditions required of different kinds of assays—such as immunoassay vs. colorimetric blood chemistry—make them inappropriate for multiplexing these different assay types. Again, centralized laboratories may achieve such integration by splitting samples and performing the assays in different devices. The centrifugal microfluidic platform with optical detection allows for a variety of assay technologies to be implemented in parallel using a single instrument and disposable.
A further significant problem of currently implemented immunofluorescent bead assays is that they are run on instruments developed for hematologic flow cytometry. While these instruments are capable of performing these assays, they are both expensive and complex, requiring significant maintenance and calibration. As a result, there exists a need for low-cost and low-maintenance instrumentation for performing such assays.
Furthermore, immunofluorescent bead assays are typically implemented using sheath flow, in which beads are diluted and aligned by the focusing flow of a buffer around the bead liquid stream. This has the dual effect of separating particles from one another such that they are not simultaneously within the detection volume of the instrument and positioning them in roughly the same place in the optical system. While this is very useful for hematologic assays, it does not confer any advantage for the detection of large numbers of essentially identical objects, such as fluorescent beads, and greatly complicates the functioning of the system. Similar statements hold if the bead sample consists of two or more well-defined populations differentiated for example by emission spectrum of their fluorescent labels.
In cases where sheath flow may be desired—extremely highly multiplexed immunoassays, or hematologic assays run on the same system as the immunoassay system—the centrifugal format provides a significant advantage over conventional pump-based systems. In the latter, typically multiple pumps are used for sample and sheath fluid flow, and great care must be taken to minimize the pulsatility of the pumps, maintaining a defined, smooth flow and ratio between sample and sheath flow. Because pumping due to centrifugal force is pulseless, such variations are inherently not present in the system.
A number of methods and devices exist for performing Bead-Based Immunoassays.
US patent publication number US2004/096867, Anderson et al, describes a system with trapped beads that are “washed through”, one weakness with this US patent publication is that it does not provide a means for moving beads from one point to another, making single bead detection impossible. A further weakness is that systems with trapped beads forming the solid phase require manufacturing techniques that are demanding. For example, in order to provide highly reproducible binding of analyte molecules and fluorescent labels, the packed ‘bed” of beads must be reproducibly structured; poor packing of the beads can lead to channels within the bead bed that pass analyte rapidly, without sufficient time for binding, resulting in low binding efficiency.
PCT Patent Publication number WO2006/110098, Gyros Patent AB, discloses a centrifugal based microfluidic device that comprises a microchannel structure in which there is a detection microcavity which in the upstream direction is attached to an inlet microconduit for transport of liquid (transport microconduit) to the detection microcavity and which is used for detecting the result of a reaction taking place in the detection microcavity or in a reaction microcavity positioned upstream of the detection microcavity. This application is primarily directed toward providing means for generating fluid “plug” flow and for joining fluids without bubbles or blockages and requires hydrophobic surface treatment for valves. The feature sizes for such structures have been realized using extensions for compact disc compression/injection moulding technology, which is incapable of producing structures which can hold larger volumes that are clinically relevant (e.g., 10 s to 100 s of uL). Discs manufactured by Gyros typically have features <=0.5 mm deep and do not operate for small or tiny volumes.
In WO9853311A2, Gamera Bioscience Corp, devices are disclosed for the performance of competitive immunoassays on a microfluidic disc. These are performed using a stationary solid phase—e.g., antibodies dried within a chamber on the disc. Furthermore, detection is via color-formation by a substrate specific for binding.
Binding of antibody directly to disc materials presents manufacturing difficulties which can be avoided by binding the antibodies instead to a “mobile” solid phase such as beads. There is no need for coating disc structures and maintaining them in the wet state for significant reaction times. Beads may also be processed in bulk quantities sufficient for hundreds of thousands of disposables under optimal conditions, including vigorous agitation to enhance reaction kinetics during their conjugation with capture antibodies.
Detection via colorimetric substrate is attractive in that it allows amplification of signal through the enzymatic reaction causing the build-up of color, but it is far more sensitive to temperature variations than is a simple fluorescent-binding assay. This temperature-sensitivity also makes storage of enzyme-based reagents for long periods (long shelf life) difficult.
In US20040089616, Kellogg et al. discloses a microfluidic disc for evaluation of glycated haemoglobin, total haemoglobin, and glucose in whole blood. One portion of the microfluidic disc uses an affinity matrix comprised of agarose beads retained between frits within the flow path of lysed, dilute blood: The glycated fraction is bound to the beads as it flows through, and the non-glycated fraction is measured photometrically in a cuvette. Combined with the measurement of total haemoglobin in another cuvette, this provides the glycated haemoglobin fraction. There are several problems to this formatting of an affinity method. First, retention of the beads requires either a) inserted elements, such as frits or b) channel constrictions in 1 dimension (“weirs”, which are too shallow for the passage of a bead) or 2 dimensions to retain the solid phase beads. In each case, manufacturing requirements are significant, requiring specialized methods for the production of features as low as a few to a few tens of microns. It is preferable to manufacture devices using more conventional methods such as the machining of injection moulding tools followed by injection moulding.
It is therefore an object of the invention to provide an immunoassay diagnostics device, system and/or method to overcome at least one of the above mentioned problems.